Temporal control of cell patterning, signaling, and movement in early embryos

果蝇胚胎背腹（DV）轴基因表达的沿着调控是果蝇胚胎发育的一个重要机制。 发展模式的范式。顺式调控元件的比较研究， 支持表达沿着DV轴从许多研究小组已经清楚地表明， 通过多种转录因子组合输入增强子， 基因表达的空间输出。这种模式化过程的关键调节器是 母亲提供的转录因子Dorsal（Dl），NF κ B的同系物。Dl功能为 形态发生素以浓度依赖性方式激活靶基因表达沿着 DV轴，有助于合子基因表达的启动， 母合子转换（MZT）。使用实时成像，我们定量了胚胎中的DI梯度， 令人惊讶的是，发现水平不仅在空间上变化，而且在时间上也会变化。我们 上一个资助期的重点是研究这些直接投资动态对目标的影响， 使用定量方法分析活体成像或固定胚胎的基因表达 时间序列数据提供洞察力。在目前的提案中，我们遵循三个新的和令人兴奋的 方向，这涉及到早期胚胎细胞行动的时间，并作为一个结果， 以前的工作。项目1涉及研究广泛表达的激活子和抑制子 在MZT过程中，它们协同控制合子基因表达的开始。我们假设 广泛表达的阻遏物与先锋激活物在控制 染色质可及性，从而也调节合子基因表达的起始。计划2 重点剖析了表达的长基因的短转录本的功能， 特别是在早期的合胞体胚胎中。我们假设这些简短的转录本 作为显性负性变体发挥作用，调节细胞信号传导途径激活的时间 信号分子。项目3的重点是确定FGF信号传导的机制， 调节粘附连接（AJs）及其与肌动蛋白细胞骨架的相互作用， 胚胎中的第一次上皮向间质转化（EMT）;特别是，了解 与FGF配体Pyramus相关的降解决定子如何限制信号传导时间。总体目标 这项研究计划的目的是了解这些细胞活动的时间- 模式、信号和运动--在发育中的果蝇胚胎中受到控制 并提供适用于高等动物的一般见解。虽然许多研究都集中在 空间输出的基因表达，较少是已知的时间动态模式。 果蝇胚胎是研究MZT的易处理系统，因为它发生在3-4小时内， 与植入前哺乳动物胚胎中的几天形成对比。果蝇胚胎是 也适合活的体内成像和跟踪分析，使其非常适合研究 新生转录和细胞形态。最后，我们建议的研究将提供一般 深入了解高等动物早期胚胎发育，因为许多调节机制可能 保守的

Regulation of gene expression along the dorsal-ventral (DV) axis of Drosophila embryos serves as a paradigm of developmental patterning. Comparative studies of cis-regulatory elements that support expression along the DV axis from many research groups have made it clear that combinatorial input into enhancers by multiple transcription factors drives distinct spatial-outputs of gene expression. A pivotal regulator of this patterning process is the maternally-provided transcription factor Dorsal (Dl), homolog of NFKB. Dl functions as a morphogen to activate target gene expression in a concentration-dependent manner along the DV axis, contributing to the initiation of zygotic gene expression at the maternal-to-zygotic transition (MZT). Using live imaging, we quantified the Dl gradient in embryos and found, surprisingly, that levels change not only in space but also build in time. Our focus during the previous funding period was to study the impact of these Dl dynamics on target gene expression using quantitative approaches involving analysis of live imaging or fixed embryo time-series data to provide insight. In the current proposal, we follow three new and exciting directions, which relate to the timing of cell actions in early embryos and arose as a result of the previous work. Project 1 involves studying how broadly-expressed activators and repressors cooperate to control the onset of zygotic gene expression during the MZT. We hypothesize that broadly-expressed repressors are equally important to pioneer activators in the control of chromatin accessibility and thereby also regulate initiation of zygotic gene expression. Project 2 focuses on dissecting the function of short-transcripts for long genes that are expressed specifically in the early syncytial embryo. We hypothesize that these short transcripts act to regulate timing of cell signaling pathway activation by functioning as dominant-negative variants of signaling molecules. Project 3 focuses on identifying the mechanism by which FGF signaling regulates adherens junctions (AJs) and their interaction with the actin cytoskeleton to contribute to the first epithelial-to-mesenchymal transition (EMT) in embryos; in particular, to understand how a degron associated with one FGF ligand, Pyramus, limits signaling time. The overarching goal of the proposed research program is to understand how the timing of these cell activities - patterning, signaling, and movement - are controlled in developing Drosophila embryos and to provide general insights applicable to higher animals. While many studies have focused on spatial outputs of gene expression, less is known about the temporal dynamics of patterning. Drosophila embryos are a tractable system to study MZT as it occurs in 3-4 hours, in contrast to taking days in preimplantation mammalian embryos. The Drosophila embryo is also amenable to live in vivo imaging and tracking analyses making it well-suited to the study of nascent transcription and cell morphology. Lastly, our proposed studies will provide general insight into early embryo development of higher animals as many regulatory mechanisms are likely conserved.